Among some 60 serologically related insect-borne flaviviruses, the four dengue virus serotypes (DEN1-4) are most important in terms of human morbidity which is estimated to involve millions of individuals every year. For this reason, the World Health Organization has assigned a high priority to the development of a dengue virus vaccine. Several new strategies for vaccine development based on the use of cloned dengue cDNA for synthesis of dengue protective antigens have yielded encouraging results. Previously, the sequence of DEN4 envelope glycoprotein (E) was analyzed by systematic carboxy-terminal deletion and expression of the resulting truncated E product in a vaccinia virus recombinant. Unlike the full-length E, 80% E was detected in high concentration on the cell surface and was secreted extracellularly. Significantly, DEN4 80%E induced a higher antibody response in mice than did the full-length E. MVA is a highly attenuated vaccinia virus deletion mutant, derived from the wild type virus by multiple serial passage in chicken embryo fibroblast cells. MVA and recombinants derived from it replicate efficiently in chick embryo fibroblast cells (CEF), but fail to replicate in human or other mammalian cells. In collaboration with Drs L. Wyatt and B Moss and B. Murphy, we employed the recently developed, highly efficient MVA expression vector to construct recombinants that contained the 80% E coding sequence of DEN4 or DEN2. Recombinant MVA-DEN2 80%E was shown to induce a higher level of antibody response in mice than recombinant MVA-DEN4 80%E. Recombinant MVA-DEN2 80%E was evaluated in monkeys for immunogenicity and protective efficacy against DEN2 challenge. Monkeys inoculated with MVA-DEN2 80%E in a two dose schedule developed a low to moderate level of DEN2 neutralizing antibodies. Two of the four inoculated monkeys that developed the highest DEN2 antibody titer were protected against subsequent DEN2 challenge. On the other hand, the other two recombinant inoculated-monkeys which developed less neutralizing antibodies, developed viremia lasting 2-3 days. Thus, partial protection against DEN2 challenge was achieved in monkeys immunized with the MVA recombinant. In a second experiment, monkeys were inoculated with MVA-DEN2 80% E in a three dose schedule to determine if a booster response in antibody could be achieved and if solid protection against challenge would result. Following inoculation with a third dose of the MVA recombinant, three of the four monkeys tested developed antibodies against DEN2 at a titer significantly higher than that detected after the second inoculation and all four monkeys were completely protected against DEN2 challenge. The results of the second study showed that monkeys responded to a triple immunization schedule with the non-replicating MVA by becoming completely resistant to homotypic dengue virus challenge. The observed safety and efficacy of the candidate vaccine are encouraging and further studies to produce MVA- recombinants expressing highly immunogenic E of the other three dengue serotypes appear to be warranted.